Methods for diagnosing pre-menstrual syndrome

ABSTRACT

A method for diagnosing premenstrual syndrome in a female human comprising the step of measuring the ratio of esterified L-carnitine to free L-carnitine in a patient&#39;s serun or plasma. Women who have an esterified to free L-carnitine ratio of greater than about 0.22 are diagnosed as having premenstrual syndrome.

TECHNICAL FIELD OF THE INVENTION

The present invention provides biochemical assays for diagnosingpremenstrual syndrome in human females. The invention also providesdiagnostic kits that utilize these assays.

BACKGROUND OF THE INVENTION

Premenstrual syndrome (PMS) has been defined as a conditioncharacterized by symptoms which recur in the late luteal phase ofsuccessive menstrual cycles, but are normally absent during menses andearly follicular phase K. Dalton et al., "PMS: The Essential Guide toTreatment Options; Thorsons, ed., London, England (1994)!. The symptomsassociated with PMS range in severity from cravings for sweet or saltyfoods to headaches and exhaustion to depression, mood swings andirritability.

In its more severe manifestation, women who suffer from PMS are unableto maintain jobs because of their cyclical anger or inability toconcentrate caused by the disease. In some individuals, PMS is so severethat it causes suicidal and homicidal feelings that, in some cases, areactually acted upon. It has been estimated that between 20 and 40percent of menstruating women suffer from PMS symptoms severe enough tointerfere with their normal daily activities D. Rovner et al.,Premenstrual Syndrome, American Council on Science and Health 1986!.

To this day, the medical community is still not universally agreed thatPMS is a medical disease. This has led to misdiagnosis and mistreatmentof various PMS symptoms as purely psychological disorders. Moreover,certain PMS symptoms may also be observed in other disease states,leading to complications and difficulties in accurate diagnosis.

The biochemical changes responsible for PMS are still uncertain.Progesterone levels have been implicated in the disease, but studiesattempting to correlate plasma progesterone levels and PMS have reachedcontrary conclusions. Some studies suggest that PMS is correlated withhigher progesterone levels K. N. Muse et al., N. Engl. J. Med, 311, pp.591-93 (1984)!, others suggest a correlation with lower progesteronelevels K. Dalton, "The Premenstrual Sydrome and Progesterone Therapy",William Heineman, ed., London, England (1984)!, still others suggest nocorrelation at all M. R. Mundy et al., Clin. Endocrinology, 14, pp. 1-9(1981)!. Despite these disparities, relief of PMS symptoms has beenreported after administration of progesterone to PMS patients.

The diagnosis of PMS is somewhat subjective because it is based on theoccurrence of cyclical symptoms during the menstrual cycle. The treatingphysician must rely upon the patient to ascertain the occurrence andcyclicity of such symptoms, usually through the patient's charting ofsuch symptoms on a daily basis for two or three menstrual cycles.Because each patient's analysis of whether or not she is suffering froma given symptom varies, it may be difficult to identify the disease inless severe cases of PMS. Moreover, in the more severe cases of PMS, thepatient may be so disabled as to be unable to accurately chart hersymptoms or unable to chart the symptoms at all.

Prior art biochemical tests for PMS are known in the art. For example,U.S. Pat. No. 5,093,265 to Portman et al., describes a test formeasuring circulating antibodies to luteinizing hormone (LH) to diagnosePMS. M. E. Dalton et al., Postgraduate Medical Journal, 57, pp. 560-61,reports a correlation between PMS and sex hormone-binding globulinconcentrations in women. Unfortunately, the accuracy and viability ofthese tests are questionable. Neither has been adopted by the medicalcommunity.

Thus, there remains a great need for an accurate biochemical test forthe diagnosis of PMS.

SUMMARY OF THE INVENTION

The present invention fills this need by providing accurate biochemicalassays for diagnosing PMS and kits which employ them. The assays andkits of this invention measure the ratio of esterified L-carnitine tofree L-carnitine in a patient's serum or plasma. Women who have anesterified:free L-carnitine ratio of greater than about 0.22 arediagnosed as having PMS.

In one aspect, the assays and kits of this invention are designed toconfirm or contrast a diagnosis of PMS based upon the occurrence ofcyclic symptoms. The assays and kits are also designed to allowphysicians to make an initial diagnosis of PMS, without the need to relyupon a patient's charting of her symptoms over a two to three menstrualcycle period. The identification of patients who have PMS even thoughthey exhibit only minor symptoms is important. Such women maypotentially experience much more severe symptoms if, for example, theyincrease the fat in their diet, if they go on a severecalorie-restricted diet or if they begin taking birth control pills orother contraceptive steroids.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of this invention, a PMS symptom is defined as any ofthe following: cravings for sweet or salty foods; cravings for alcohol;compulsive eating; bloating; diarrhea or constipation; nausea; weightgain or loss; tickling, crawling or prickling on the skin; cold sores;pimples or acne; styes; sinus problems; easy bruising; backache; jointpain or arthritis; overall body aches; cramps; headache; exhaustion;angry outbursts; poor concentration; difficulty in making decisions;insomnia; mood swings; suicidal feelings; crying spells; depression;feelings of worthlessness; easy irritability; feelings of loneliness;frequent accidents or near accidents; panic attacks; breast pain;swelling of fingers, ankles or face; heart palpitations; dizziness;asthma or wheezing; fainting; chest pain; and sensations of tremoring.

In addition, a PMS symptom must exhibit cyclic recurrence at similartimes, excluding periods of menstrual flow, during two to threeconsecutive menstrual cycles. In order to determine whether a conditionis cyclic, a female should chart each of these conditions every day forat least two consecutive menstrual cycles. In addition, the femaleshould also chart the beginning and end of menstrual flow during thesecycles. After charting such conditions through two cycles, both thefemale and the practitioner can determine whether the occurrence of anyparticular condition during the next cycle qualifies as a PMS symptom.

The present invention provides methods for diagnosing PMS in women bydetermining the serum or plasma esterified L-carnitine:free L-carnitineratio. At levels above about 0.22, PMS is indicated.

The basis of this invention is the inventors' hypothesis that a defectin fat metabolism is responsible for the plethora of cyclic symptomsthat characterize PMS. More specifically, the inventors believe that PMSis caused by a defect in the translocation of fatty acids into themitochondria.

Fat is metabolized in the mitochondria of cells. Fatty acids areinitially activated outside the mitochondria by thioester linkage tocoenzyme A (CoA). The resulting fatty acid-CoA ester then reacts withL-carnitine in a transesterification reaction catalyzed by an outermitochondrial membrane enzyme, carnitine palmitoyltransferase, producingacyl-carnitine. The acyl carnitine is then translocated across themitochondrial membrane where it is transesterified with CoA at the innermitochondrial membrane to regenerate acyl-CoA and free carnitine.

Certain mutations in genes encoding enzymes that play a role, eitherdirectly or indirectly, in L-carnitine-mediated translocation of fattyacids can result in a build-up of esterified carnitine, thus increasingthe esterified:free L-carnitine ratio. The resulting build-up ofunmetabolized fatty acids in the cell alters normal cellular functionand causes the physical and mental effects that are characteristic ofPMS.

Without being bound by theory, applicants believe that women who sufferfrom PMS may possess a genetic mutation in the transcription controlregion of one or more of the genes necessary for fat metabolism. Morespecifically, applicants believe that this mutation is in aprogesterone-responsive enhancer element--a portion of a gene capable ofaltering transcription levels through the binding of progesterone. Thiswould explain the efficacy of progesterone in alleviating PMS symptoms.

Thus, according to one embodiment, the invention provides a method ofdiagnosing premenstrual syndrome in a female human comprising the stepsof:

removing an aliquot of blood from said female human on a day when saidfemale human is suffering from at least one PMS symptom and isolatingthe serum or plasma from said blood;

determining the ratio of esterified L-carnitine to free L-carnitine insaid serum or plasma; and

diagnosing said female human as suffering from premenstrual syndrome ifsaid ratio of esterified L-carnitine to free L-carnitine is greater thanabout 0.22 and total L-carnitine is greater than 20 μmoles/liter.

In addition, the female human must refrain from taking supplementalL-carnitine for at least fifteen day prior to removing an aliquot ofblood for use in this assay. In addition, the female must fast for atleast 8 hours prior to removing blood for the assay. Both of theserequirements are necessary to insure obtaining accurate concentrationsof free and esterified L-carnitine.

In this embodiment, the occurrence of any single PMS symptom issufficient for indicating a day on which ratio of esterified L-carnitineto free L-carnitine may be determined. Preferably, the PMS symptom is atleast one of the following: cravings for sweet or salty foods;compulsive eating; diarrhea or constipation; weight gain or loss;tickling, crawling or prickling on the skin; cold sores; sinus problems;overall body aches; headache; exhaustion; angry outbursts; mood swings;crying spells; depression; easy irritability; frequent accidents or nearaccidents; breast pain; and swelling of fingers, ankles or face. Morepreferably, the female will be suffering from at least three PMSsymptoms, at least one of which is selected from the above preferredlist.

The amount of serum or plasma required for a single assay is about 0.6ml. Typically, about 5 ml of blood is collected from the patient into aheparin-coated glass tube. Preferably, the blood is collected using aVacutainer collection device using a standard heparinized collectionvessel.

Once removed, the blood or the plasma or serum derived therefrom may beassayed immediately or stored for a period of time prior to performingsaid assay. Although the blood, serum or plasma may be stored at roomtemperature for up to 24 hours, it is preferable that it be stored at 4°C. if the assay is not performed immediately after it is obtained fromthe female. If the blood, plasma or serum will not assayed within 3days, it is further preferred that it be stored frozen at -20° C. Atthis temperature, it has a shelf life of approximately 4 weeks and maybe assayed at any time during that time frame. Preferably, serum orplasma will be separated from the red blood cells immediately followingblood collection. The serum or plasma may then be stored as indicatedabove or assayed directly.

The determination of the ratio of esterified L-carnitine:freeL-carnitine may be achieved by any combination of assays which arecapable of determining at least two the following three parameters: freeL-carnitine concentration, esterified L-carnitine concentration or total(free+esterified) L-carnitine concentration. The majority of knownassays are incapable of measuring esterified L-carnitine concentrationsdirectly in the presence of free L-carnitine. Accordingly, the preferredassays of this invention measure total L-carnitine and free L-carnitineand calculate esterified L-carnitine as the difference between total andfree L-carnitine.

In general, the assays for total L-carnitine are merely assay for freeL-carnitine preceded by a hydrolysis step that converts any esterifiedL-carnitine to free L-carnitine. This hydrolysis step is preferablyachieved by heating the serum or plasma sample under alkaline pHconditions. Alternatively, hydrolysis may achieved through the use of anacyl-carnitine esterase, such as described in U.S. Pat. No. 5,385,829 toTakahashi et al.

Known assay methods for carnitine which may be employed in thisinvention fall into several different groups. The first type of assayreacts L-carnitine in a sample with acetyl CoA in the presence ofcarnitine acetyl transferase (CAT) to produce acetyl-L-carnitine andCoA. The CoA is then reacted with 5,5'-dithio-bis-2-nitrobenzoate (DTNB)to generate thiophenylate ion which is calorimetrically measured. N.Marquis et al., J. Lipid Res., 5, pp. 184-87 (1964); and I. Fritz etal., J. Biol. Chem., 238, pp. 2509-17 (1963). An automated version ofthis assay is described in U.S. Pat. No. 5,316,917 to Roe.

The second type of assay reacts L-carnitine in a sample with NAD in thepresence of L-carnitine dehydrogenase to produce 3-dehydrocarnitine andNADH. The UV absorption of NADH is followed spectrophotometrically todetermine L-carnitine concentrations. Z. Fresenius, Anal. Chem., 320,pp. 285-89 (1985); Schopp et al., Eur. J. Biochem., 10, pp. 56-60(1969); H. Aurich et al., Eur. J. Biochem., 6, pp. 196-201 (1968). Amodified version of this assay is also described in U.S. Pat. No.5,266,463 to Takahashi et al.

The third type of assay is similar to the DTNB method described above,but replaces DNTB with n- p-(2-benzimidazoyl)-phenyl!-malimide (BIPM).The fluorescence of the resulting CoA-BIPM is then measured. K. Watanabeet al., Ann. Rep. MHW Institute for Nerve Disease, pp. 315-18 (1986).

The final and most preferred type of assay reacts L-carnitine in asample with radioactively labeled (³ H- or ¹⁴ C-) acetyl CoA in thepresence of CAT. This produces radiolabelled acetyl-L-carnitine which,after removal of any excess labeled acetyl CoA, is quantitated bymeasuring radioactivity. M. Hamamoto et al., J. Japan. Nut. Food Soc.,41, pp. 389-95 (1988); R. Parvin et al., Anal. Biochem., 79, pp. 190-201(1977); J. McGarry et al., J. Lipid Res., 17, pp. 277-81 (1976); G.Cederblad et al., Clin. Chim. Acta, 37, pp. 235-43 (1972).

Known methods of determining concentrations of esterified L-carnitinemay also be employed in the assays of this invention, but are lesspreferred. These methods typically involve hydrolysis ofacyl-L-carnitines in a sample followed by quantification of the releasedfatty acids using chromatography. See U.S. Pat. No. 5,385,829.

According to the most preferred embodiment, free L-carnitine is assayedby reacting a first portion of human female serum with ¹⁴ C-acetyl-CoAin the presence of carnitine acetyl transferase and N-ethylmalemide toform ¹⁴ C-acetyl-carnitine;

employing means to remove any unreacted ¹⁴ C-acetyl-CoA; and

determining the amount of ¹⁴ C-acetyl-carnitine formed by countingradioactivity and comparing said counts to a standard curve.

Means for removing ¹⁴ C-acetyl-CoA without removing ¹⁴C-acetyl-carnitine are known in the art and include the use of activatedcharcoal or an anion exchange resin. Preferably an anion exchange resinis used. Most preferably, removal is achieved through a column of Dowexanion exchange resin.

Total L-carnitine is preferably assayed by heating a second portion ofhuman female serum, preferably to about 37° C., for 10-30 minute underalkaline pH conditions. Alkaline pH conditions are preferably obtainedby treating the serum with 0.5 volumes of 1.5M KOH. After heat/alkalinetreatment, the serum is cooled to room temperature, adjusted to pH 7.6,preferably with H₃ PO₄, and assayed for free carnitine as describedabove.

In each assay, the radiolabelled ¹⁴ C-acetyl-carnitine is counted usingany commercially available scintillation counter. The counts in thesample are then compared to a standard curve derived from knownconcentrations of free L-carnitine treated under the same assayconditions.

The results of any of these assays may then be used to diagnose PMS. Ifthe total L-carnitine concentration is above 20 μM and the ratio ofesterified: free L-carnitine is above 0.22, the patient is diagnosed assuffering from PMS. Total L-carnitine levels below 20 μM indicate acarnitine deficiency that would cause skewed esterified:free L-carnitineratios. This, in turn, would interfere with the accuracy of thediagnosis. Normal total plasma L-carnitine levels range from 25-79 μM.

In order to ensure accurate diagnosis, any ratios between 0.20 and 0.25should be confirmed by repeating the assay on a sample of blood taken ona different day where the female human is suffering from PMS symptoms.

This assay is useful to confirm a preliminary diagnosis of PMS basedupon the occurrence of PMS symptoms. If the female human does not have aratio above the 0.22 threshold, some other disorder, either physical ormental, is responsible for her PMS symptoms.

According to an alternate embodiment of the invention, the assaysdescribed above can be used to screen any ovulating human female forPMS. The advantage of this embodiment is that it does not require orrely upon the charting or occurrence of PMS symptoms.

In this embodiment, a sample of blood is taken from a female human onany day that she is not experiencing menstrual flow. Again, the womanmust have fasted for at least 8 hours prior to the taking of blood. Inaddition, the female must not have taken any L-carnitine supplements inthe past 15 days. The assay is run on serum or plasma isolated from theblood as described above and a ratio of esterified:free L-carnitine isobtained. If the ratio is less than about 0.22, the assay is repeated upto 3 more times at 7 day intervals, unless one of the intervals occurson a day when the female is experiencing menstrual flow. If one of theintervals should fall on such a day, blood should not be taken until thefirst day after flow has ceased. If additional assays are needed, thenext seven day interval is calculated from the day that blood wasactually taken, not from the day when blood was supposed to be taken,but was not because the female was experiencing menstrual flow.

If on any of the four intervals, the ratio of esterified:freeL-carnitine is above about 0.22, the woman is diagnosed as sufferingfrom PMS and no additional assays need be performed. If the ratio isbelow about 0.22 after the four assays, the women is diagnosed as freefrom PMS.

According to an alternate embodiment, the invention provides anothermethod for using esterified:free L-carnitine ratios to diagnose PMS inan ovulating human female without requiring the charting of PMSsymptoms. This method comprises the steps of:

determining the length of a human female's menstrual cycle;

removing an aliquot of blood from said human female 5 days prior to theend of a menstrual cycle and isolating the serum or plasma from saidblood; and

determining the ratio of said esterified L-carnitine to free L-carnitinein said serum or plasma;

If the ratio is less than about 0.22, the assay is repeated every dayfor up to four more days until said ratio of esterified L-carnitine tofree L-carnitine is greater than about 0.22 and said amount of totalL-carnitine is greater than 20 μmoles/liter.

If the ratio on any day is greater than about 0.22, the female human isdiagnosed as suffering from premenstrual syndrome and need not be testedon subsequent days. If the ratio is less than about 0.22 on all fivedays (i.e., 5, 4, 3, 2 and 1 day before the end of the menstrual cycle),then the woman is diagnosed as being free from premenstrual syndrome.

The determination of a woman's menstrual cycle length can be made bysimply calculating the interval between the beginning of menses in anygiven cycle and the next recurrence of menses. Many women will, in factbe able to calculate menstrual cycle length based upon their ownprevious observation. For example, many women routinely record the firstday of menses in a calendar book. The length of a woman's menstrualcycle does not vary significantly from one cycle to the next. Therefore,calculations of menstrual cycle length based upon prior observation issufficient for this assay.

As a practical matter, once the menstrual cycle length is determined,five days prior to the end of the menstrual cycle is determined bycounting (menstrual cycle length--5) days after the onset of menses.

The removal of blood and determination of free and esterifiedL-carnitine for this assay is performed as described above.

As a practical matter, a portion of the blood removed from the female inany of the above-described assays should be subjected to a routine bloodwork-up, such as that typically performed during an annual physicalexamination. This will rule out any abnormalities that may cause falsepositives in this assay. Such abnormalities include severe amino aciddeficiencies and vitamin B₁₂ deficiency, as well as any conditions whichcause severe lactic acidosis.

According to another embodiment, the invention provides a kit fordiagnosing PMS in a female human comprising:

means for determining the concentration of free L-carnitine in bloodserum or plasma;

means for determining the concentration of esterified L-carnitine inblood serum or plasma; and

instructions for using the elements of the kit to obtain a ratio ofesterified L-carnitine:free L-carnitine in said serum or plasma and forcorrelating said ratio to a diagnosis of PMS.

The components of the kit are typically contained within a singlepackage.

Any of the means for determining free and esterified L-carnitineconcentrations described above may be employed in the kits of thisinvention. Preferably the means for determining concentration of freeL-carnitine in blood serum or plasma comprise a first containercontaining a solution of acetyl CoA at a concentration of between 1.2and 120 mM and a second container containing a solution of DTNB or BIPMat a concentration of between 0.27 to 27 mM at a pH of between about6.5-8.5. The solutions in the first and second containers are mixedtogether prior to use to form a solution comprising 0.23 to 23 mM DTNBor BIPM and 0.17 to 17 mM acetyl CoA. More preferably, the means fordetermining concentration of free L-carnitine additionally comprises athird container containing a solution of carnitine acyl transferase at aconcentration of between 1.72 and 172 kU/liter.

The means for determining the concentration of esterified L-carnitinepreferably comprises a first container containing a solution of KOH at aconcentration of between 1.5 and 5M or an acyl carnitine esterase at aconcentration of between 0.1 to 10 kU/liter. This component converts theesterified L-carnitine in the sample to free L-carnitine. The resultingtotal L-carnitine (free+de-esterified) is then quantified. EsterifiedL-carnitine is quantified by subtracting free L-carnitine concentrationfrom total L-carnitine concentration.

The instructions included in the kits of this invention are typically ona printed sheet contained within the package or written on the packageitself. Of course, the instructions may be included with the kit byother means, such as on a computer readable form (i.e., diskette) in thekit.

The instructions detail the use of the elements of the kit for carryingout the assay and for obtaining a ratio of esterified L-carnitine:freeL-carnitine, as well as how to interpret the results in order todiagnose PMS.

According to an alternate embodiment, the kit may additionally compriseany or all of the following: a container containing a solution ofL-carnitine at a concentration of between 0.1 and 10 mM for use as astandard; a container containing a solution of octanoyl-L-carnitine at aconcentration of between about 0.1 and 10 mM for use as anesterified-L-carnitine control; and a container containing a solution ofH₃ PO₄ or 3- N-morpholino!propane sulfonic acid in HCl at aconcentration of between about 0.1 and 10M for neutralizing the serumafter hydrolysis of esterified L-carnitine in KOH.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLE 1

Approximately 70 women identified as suffering from PMS symptoms on thebasis of symptom charting over two to three menstrual periods were usedin the initial study. A sample of blood (5 ml) was removed from each ofthese women on day 21 of their menstrual cycle. (Day 1 is defined as thefirst day of heavy menstrual flow.) The blood was allowed to clot and weisolated the serum. The serum was stored at 4° C. for 48 hours prior toperforming the assay.

The serum was then assayed for free and total L-carnitine concentrationsessentially using the ¹⁴ C-acetyl CoA method described by R. Parvin etal., Anal. Biochem., 79, pp. 190-201 (1979). The method used differedfrom the published method in that a Dowex anion exchange column, ratherthan charcoal, was used to remove unreacted ¹⁴ C-acetyl CoA from thesample prior to scintillation counting.

Approximately 90% of the women displayed an esterified L-carnitine:freeL-carnitine ratio of greater than 0.22. The results of the assay werethen correlated with the presence of PMS symptoms on the day the sampleswere taken. Surprisingly, there was an almost 100% correlation betweenratios below 0.22 and the absence of PMS symptoms.

This result suggested that women suffering from PMS may display a ratiobelow the 0.22 threshold on days when they are not experiencingsymptoms. Therefore, we decided to take blood samples on a day when awoman was displaying PMS symptoms, rather than on a specific day of themenstrual cycle.

EXAMPLE 2

Approximately 40 women identified as suffering from PMS symptoms on thebasis of symptom charting over two to three menstrual periods were usedin the next study. These women had not taken any L-carnitine supplementsin the past 15 days. In addition, these women had fasted for at least 8hours prior to having blood drawn. A sample of blood (5 ml) was removedfrom each of these women on a day when she was suffering from PMSsymptoms between day 19 and 25 of their menstrual cycle. The blood wasallowed to clot and we isolated the serum. The serum was stored at 4° C.for 48 hours prior to performing the assay. The assay was performed asdescribed in Example 1.

Each of the women had an esterified L-carnitine:free L-carnitine ratioof greater than 0.22. Control women who do not experience any PMSsymptoms all have ratios of less than 0.22. These results demonstratedthat a diagnosis of PMS based upon charting of symptoms can be confirmedby a blood esterified L-carnitine:free L-carnitine ratio of greater than0.22 on a day when a woman is suffering from such symptoms.

EXAMPLE 3

100 random women are chosen for the next study. These women have nottaken any L-carnitine supplements in the past 15 days. In addition,these women have fasted for at least 8 hours prior to having blooddrawn. A sample of blood (5 ml) is removed from each of these women on aday when they are not experiencing menstrual flow. The blood iscollected by use of a Vacutainer device into a heparin-coated glassvial. The blood is centrifuged and the plasma is isolated. Free andtotal L-carnitine are measured as described in Example 1. Women who havean esterified:free L-carnitine ratio of greater than 0.22 are diagnosedas having PMS.

Seven days later, a second sample of blood (5 ml) is removed from anywomen whose esterified:free L-carnitine ratio is below the 0.22threshold in the first assay. Women who are experiencing menstrual flowat the time of the second 7 day interval are not tested until flow hasceased. Again, plasma is isolated and esterified:free L-carnitine ratiosare measured. Women who have an esterified:free L-carnitine ratio ofgreater than 0.22 in the second assay are diagnosed as having PMS.

Women who are not diagnosed as having PMS in the prior two assays have athird sample of blood removed 7 days after the previous sample, or, ifthey are experiencing menstrual flow on that day, the first day thatflow ceases. Plasma from the blood is assayed in the same manner. Womenwho have an esterified:free L-carnitine ratio of greater than 0.22 inthe third assay are diagnosed as having PMS.

The procedure is repeated a fourth time on any women not diagnosed ashaving PMS by the three previous assays. Any woman who has anesterified:free L-carnitine ratio of less than 0.22 in all four assaysis diagnosed as being free from PMS.

The results of this assay are then confirmed by the PMS symptom chartingmethod. Women diagnosed as having PMS by the L-carnitine assay displayPMS symptoms over a two to three menstrual cycle charting period. Womendiagnosed as free from PMS by the L-carnitine assay do not display PMSsymptoms over a two to three menstrual cycle charting period.

EXAMPLE 4

The menstrual cycle length (M) for each of 100 random women isdetermined based on the interval between two consecutive onset ofmenses.

A sample of blood (5 ml) is removed from each of these women on day(M)-5 following the onset of menstrual flow. These women have not takenany L-carnitine supplements in the past 15 days. In addition, thesewomen have fasted for at least 8 hours prior to having blood drawn. Theblood is collected by use of a Vacutainer device into a heparin-coatedglass vial. The blood is centrifuged and the plasma is isolated. Freeand total L-carnitine are measured as described in Example 1. Women whohave an esterified:free L-carnitine ratio of greater than 0.22 arediagnosed as having PMS.

The next day, a second sample of blood (5 ml) is removed from any womenwhose esterified:free L-carnitine ratio is below the 0.22 threshold inthe first assay. Again, plasma is isolated and esterified:freeL-carnitine ratios are measured. Women who have an esterified:freeL-carnitine ratio of greater than 0.22 in the second assay are diagnosedas having PMS.

Women who are not diagnosed as having PMS in the prior two assays have athird sample of blood removed the following day. Plasma from the bloodis assayed in the same manner. Women who have an esterified:freeL-carnitine ratio of greater than 0.22 in the third assay are diagnosedas having PMS.

The procedure is repeated the next day on any women not diagnosed ashaving PMS by the three previous assays. Again, women who have anesterified:free L-carnitine ratio of greater than 0.22 in the thirdassay are diagnosed as having PMS.

Any woman not diagnosed as having PMS from the previous four assays issubjected to another assay on the next day. Women who have anesterified:free L-carnitine ratio of less than 0.22 in all five assaysare diagnosed as being free from PMS.

The results of this assay are then confirmed by the PMS symptom chartingmethod. Women diagnosed as having PMS by the L-carnitine assay displayPMS symptoms over a two to three menstrual cycle charting period. Womendiagnosed as free from PMS by the L-carnitine assay do not display PMSsymptoms over a two to three menstrual cycle charting period.

While I have hereinbefore presented a number of embodiments of thisinvention, it is apparent that my basic construction can be altered toprovide other embodiments which utilize the methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the claims appended hereto rather than the specificembodiments which have been presented hereinbefore by way of example.

We claim:
 1. A method of diagnosing premenstrual syndrome in a femalehuman who has fasted for at least 8 hours and has not taken L-carnitinesupplements in the past 15 days, said method comprising the steps of:a.removing an aliquot of blood from said female human on a day when saidfemale human is suffering from at least one PMS symptom and isolatingserum or plasma from said blood; b. determining total L-carnitineconcentration and a ratio of esterified L-carnitine to free L-carnitinein said serum or plasma, wherein said ratio is determined by the stepsof:i. determining free L-carnitine concentration in a first portion ofsaid serum or plasma; ii. determining total L-carnitine concentration ina second portion of said serum or plasma; and iii. subtracting said freeL-carnitine concentration from said total L-carnitine concentration toobtain esterified L-carnitine concentration in said serum or plasma; andiv. dividing said esterified L-carnitine concentration obtained in stepiii by said free L-carnitine concentration obtained in step i; and c.diagnosing said female human as suffering from premenstrual syndrome ifsaid ratio of esterified L-carnitine to free L-carnitine is greater thanabout 0.22 and said total L-carnitine concentration is greater than 20μmoles/liter.
 2. The method according to claim 1, wherein at least oneof said PMS symptoms is selected from the group consisting of cravingfor sweet or salty foods; craving for alcohol; compulsive eating;bloating; diarrhea or constipation; nausea; weight gain or loss;tickling, crawling or prickling on the skin; cold sores; pimples oracne; styes; sinus problems; bruising; backache; joint pain orarthritis; body aches; cramps; headache; angry outbursts; insomnia; moodswings; suicidal feelings; crying spells; depression; feelings ofworthlessness; irritability; feelings of loneliness; panic attacks;breast pain; swelling of fingers, ankles or face; heart palpitations;dizziness; asthma or wheezing; fainting; chest pain; or sensations oftremoring.
 3. The method according to claim 2, wherein said female humanis suffering from at least three of said PMS symptoms.
 4. A method ofdiagnosing premenstrual syndrome in a female human who has fasted for atleast 8 hours and has not taken L-carnitine supplements in the past 15days, said method comprising the steps of:a. removing an aliquot ofblood from said female human and isolating serum or plasma from saidblood on a day when said female human is not experiencing menstrualflow; b. determining total L-carnitine concentration and a ratio ofesterified L-carnitine to free L-carnitine in said serum or plasma,wherein said ratio is determined by the steps of:i. determining freeL-carnitine concentration in a first portion of said serum or plasma;ii. determining total L-carnitine concentration in a second portion ofsaid serum or plasma; and iii. subtractinag said free L-carnitineconcentration from said total L-carnitine concentration to obtainesterified L-carnitine concentration in said serum or plasma; and iv.dividing said esterified L-carnitine concentration obtained in step iiiby said free L-carnitine concentration obtained in step i; and c.repeating steps a. through b. up to three more times at seven dayintervals until said ratio of esterified L-carnitine to free L-carnitineis greater than about 0.22 and said total L-carnitine concentration isgreater than 20 μmoles/liter, with the proviso that if any of said sevenday intervals occurs on a day when said female human is experiencingmenstrual flow, delaying steps a. through d. until said menstrual flowhas ceased; and d. diagnosing said female human as:i. suffering frompremenstrual syndrome if said ratio of esterified L-carnitine to freeL-carnitine at any of said seven day intervals is greater than about0.22 and said total L-carnitine concentration is greater than 20μmoles/liter; or ii. free from premenstrual syndrome if said ratio ofesterified L-carnitine to free L-carnitine at each of four consecutiveseven day intervals is less than about 0.22 and said total L-carnitineconcentration at each of said interval is greater than 20 μmoles/liter.5. A method of diagnosing premenstrual syndrome in a female human whohas fasted for at least 8 hours and has not taken L-carnitinesupplements in the past 15 days, said method comprising the steps of:a.determining the duration of said female human's menstrual cycle; b.removing an aliquot of blood from said female human 5 days prior to theend of the menstrual cycle and isolating serum or plasma from saidblood; c. determining total L-carnitine concentration and a ratio ofesterified L-carnitine to free L-carnitine in said serum or plasma,wherein said ratio is determined by the steps of:i. determining freeL-carnitine concentration in a first portion of said serum or plasma;ii. determining total L-carnitine concentration in a second portion ofsaid serum or plasma; and iii. subtracting said free L-carnitineconcentration from said total L-caraitine concentration to obtainesterified L-carnitine concentration in said serum or plasma; and iv.dividing said esterified L-carnitine concentration obtained in step iiiby said free L-carnitine concentration obtained in step i; and d.repeating steps b. through c. every day for up to four more days untilsaid ratio of esterified L-carnitine to free L-carnitine is greater thanabout 0.22 and said total L-carnitine concentration is greater than 20μmoles/liter; and e. diagnosing said female human as:i. suffering frompremenstrual syndrome if said ratio of esterified L-carnitine to freeL-carnitine on any of said days is greater than about 0.22 and saidtotal L-carnitine concentration is greater than 20 μmoles/liter; or ii.free from premenstrual syndrome if said ratio of esterified L-carnitineto free L-carnitine on each of five consecutive days is less than about0.22 and said total L-carnitine concentration on each day is greaterthan 20 μmoles/liter.
 6. The method according to claim 1, 4 or 5,wherein the free L-carnitine concentration is determined by the stepsof:a. reacting said serum or plasma with ¹⁴ C-acetyl-CoA in the presenceof carnitine acetyl transferase and N-ethylmalemide to form an amount of¹⁴ C-acetyl-carnitine; b. employing means to remove any unreacted ¹⁴C-acetyl-CoA; c. determining the amount of ¹⁴ C-acetyl-carnitine formedby counting radioactivity and comparing said counts to a standard curve.7. The method according to claim 1, 4 or 5, wherein the totalL-carnitine concentration is determined by the steps of:a. heating saidserum or plasma under alkaline pH conditions to hydrolyze any esterifiedL-carnitine; b. neutralizing the pH of said serum or plasma; c. reactingsaid serum or plasma with ¹⁴ C-acetyl-CoA in the presence of carnitineacetyl transferase and N-ethylmalemide to form an amount of ¹⁴C-acetyl-carnitine; d. employing means to remove any unreacted ¹⁴C-acetyl-CoA; e. determining the amount of ¹⁴ C-acetyl-carnitine formedby counting radioactivity and comparing said counts to a standard curve.